Free Rod. Res. Cornrns.. Volr. 12-13. pp. 41-52

Q 1991 Hanvaod Academic Publishen GmbH

Printed in the United Kingdom

Reprints available directly from the publisher Photocopying permitted by license only


Free Radic Res Downloaded from by Chulalongkorn University on 12/31/14 For personal use only.

ANTHONY J. KETTLE* and CHRISTINE C. WINTERBOURN Department of Pathology, Christchrtrch School of Medicine, Christchurch Hospital, Christchurch, New Zealand Human neutrophils stimulated with opsonized zyrnosan promoted hypochlorous acid (H0CI)-dependent loss of monochlorodimedon. Formation of HOCl was completely inhibited by catalase, and it was also inhibited up to 70% by SOD.There was no inhibition by desferal, DTPA,mannitol or dimethylsulphoxide. which excluded the involvement of *OH. Our results indicate that generation of 0; by neutrophils enables these cells to enhance their production of HOCI.Furthermore, inhibition of neutrophil processes by SOD and catalase does not necessarily implicate -OH. We propose that 0; may potentiate oxidant damage at inflammatory sites by boosting the rnyeloperoxidase-dependent production of HOCl

KEY WORDS: Myeloperoxidase, superoxide, hypochlorous acid, neutrophils

INTRODUCTION Hypochlorous acid (HOCI) is a potent oxidant discharged by stimulated neutrophils. It reacts readily with biological molecules, inactivating a,-proteinase inhibitor, other neutrophil enzymes and inflammatory molecules, and is cytotoxic to a wide variety of mammalian cells.' Since HOCl can account for at least 30% of the 0; generated during the respiratory burst,',' it may be responsible for the majority of oxidant damage mediated by neutrophils. Myeloperoxidase, the most abundant neutrophil protein, catalyses the production of HOCl from H,O, and CI-.4 H,Ozis supplied as a secondary product by the respiratory burst through the dismutation of 0; Howerver, 0; also reacts directly with myeloperoxidase6 and modulates the chlorinating activity of the purified en~yrne.'.~Under conditions where compound II accumulates, 0; can treble the production of HOCl by reducing compound II back to active ferric myeloperoxidase.' Although this mechanism has been demonstrated with the purified enzyme,'.'' there is no evidence from previous ~ t u d i e s ' ~that ~ ~ it' ~operates in the neutrophil. We have examined in detail the effect of Or on HOCl production by neutrophils" and in this presentation we report our major findings.



Neutrophils were isolated from the blood of healthy donors by Ficoll-Hypaque 'To whom correspondencc and rcprint requcsts should bc addrcsscd 47


A.J. K E l T L E AND C.C. W I N T E R B O U R N

centrifugation, dextran sedimentation, and hypotonic lysis of contaminating red cells.12 Myeloperoxidase was purified from human neutrophils to a purity index (A430/A280)of > 0.7, and its concentration was determined using Q,, = 9 I , 000 M-' crn-'.I3 Zymosan was opsonized in 30% human plasma with end over end rotation for 30 minutes at 37' C. Superoxide disrnutase (SOD), catalase, monochlorodimedon (MCD) and zymosan were purchased from Sigma. All other chemicals were of the highest grade available.

Free Radic Res Downloaded from by Chulalongkorn University on 12/31/14 For personal use only.

Methods Reactions were carried out in PBS containing I mM CaCI,, 0.5 mM MgCI, and I mg ml-' of glucose. When incubations were carried out in the absence of CI-, the buffer was 10mM phosphate, pH 7.5, containing l.OmMMgSO,, 47mM Na2S0, and 1 mgml-' of glucose. Neutrophils were incubated in a total volume of 1 ml in 1.5 ml sealed microfuge tubes with end over end rotation for 15 minutes at 37OC. At the end of this period tubes were placed in melting ice for 10 minutes and centrifuged to pellet neutrophils and zymosan. The production of HOCl was based on the loss of MCD (qW 19,000 M-'cm-').I4 0; production was measured as SOD-inhibitable cytochrome c reduction (eSM (r&&,xidiEd) 21,100 M-' cm-I).

RESULTS Eflects of superoxide dismirfase and catalase on fhe loss of M C D When neutrophils were stimulated with opsonized zymosan they promoted the loss of MCD. Catalase and SOD inhibited the loss of MCD by 93% and 73% respectively (Figure 1). The effects of these proteins were due to their enzymatic activity since the heat-inactivated enzymes and bovine serum albumin were ineffective. The loss of MCD in the absence of SOD was directly proportional to the production of 0;(Figure 2), which was used as a measure of total oxidant (0; + H202) production. In the presence of SOD the loss of MCD plateaued, so that SOD gave greatest inhibition at the highest rates of 0; production but did not inhibit at lower

FIGURE I. The effects of catalase and superoxide dismutase on the loss of M C D mediated by neutrophils stimulated with opsonized zymosan. Neutrophils (4 x 106ml-') were incubated with 5rngml-' of opsonized zymosan and 70pM M C D in total volume of I ml at 37OC Tor I5 min. Concentrations ofcatalase (CAT), SODand bovineserulnalbumin(BSA) were IOOpgml-'. 2Opgiiil-' and IOOpgml-', respectively. Enzymes were heat inactivated (H.I.) by boiling for I5 min. Superoxide production was 170pM in I5 min. Values represent meeans and ranges of duplicate expcriments.




n 0



25. 20



0 Free Radic Res Downloaded from by Chulalongkorn University on 12/31/14 For personal use only.







FIGURE 2. The effect of superoxide production on the loss of MCD mediated by neutrophils stimulated with opsonizcd zymosan. Conditions were as described in Figure I except the concentration of opsonized zymosan was varied between 0 and 5 m g m l - ' in the presence (A) or absence ( 0 )of 20pgml-' of SOD. Superoxide produced during I5 min of incubation was measured in parallel experiments by the cytochrome c assay. Data are from experiments with several neutrophil preparations.

rates. SOD did not affect the release of myeloperoxidase." Thus, it can be concluded that in the presence of SOD the increased production of HzOzeventually saturated the activity of myeloperoxidase. However, in the absence of SOD, 0; was responsible for maintaining the activity of myeloperoxidase so that H 2 0 2was efficiently converted to HOCI. Effects of oxidant inhibitors and scavengers on the loss of MCD Inhibition by catalase and SOD could indicate that the loss of MCD was due to .OH produced by the 0;-driven Fenton reaction.'' To distinguish between HOCl and .OH as the neutrophil oxidant responsible for the loss of MCD, reactions were carried out in the presence of either exogenous myeloperoxidase, or inhibitors of HOCl or -OH production. Loss of MCD was completely suppressed by the heme TABLE I The effect of myeloperoxidase and oxidant inhibitors on the loss of MCD mediated by neutrophils stimulated with opsonized zymosan. Conditions were as described in Figure I. Superoxide production was I12pM/15 min in the presence of CI- and 67pM/15 min in its absence. Conditions

MCD Loss (pM)


PMN + MCD + OZ + 250nM MPO + IOOpM azide + I mM cyanide - c1+20mM Me,SO + 40mM Me,SO + 5 m M mannitol + IOmM rnannitol + IOOpM desferal + IOOpMDTPA + IOOpM methionine

20.5 f 1.1 (3) 35.7 f 1.9 (4) 0.4 f 1.1 (4) 0.1 f 1 . 1 (4) 2.6 f 0.9 (4) 22.7 f 1.2 (2) 22.7 f 1.2 (2) 20.0 f 2.6 (2) 22.1 1 . 1 (2) 20.4 f 1.0 (2) 20.2 f 2.2 (4) 2.7 f 1.1 (4)



+ 74 - 98

- loo - ao*

+ 10 +I1 -2

+a -I +I - a7

*Corrected for decreased 0; production. Values represcnt means and standard deviations of experiments.


Free Radic Res Downloaded from by Chulalongkorn University on 12/31/14 For personal use only.



enzyme inhibitors azide and cyanide, and was enhanced by adding exogenous myeloperoxidase (Table I). It was also suppressed when cells were suspended in CI--free buffer to prevent production of HOCI. Under these conditions, the cells produced only approx. 60% of the 0; generated in CI--buffer. This in itself would result in a corresponding decrease in oxidant production. However, even when this decrease was accounted for, loss of MCD was inhibited by 80%. C!--free buffer had no effect on the amount of myeloperoxidase released.'' Dimethyl-sulfoxide (Me,SO) and mannitol react rapidly with .OH and are commonly used as -OHscavengers. However, they failed to inhibit the loss of MCD (Table I). In addition, there was no inhibition by desferal or diethylenetriaminepentaacetic acid (DTPA), both of which complex iron in a form that is unable to catalyse the 0;-driven Fenton reaction.I5 Methionine, which reacts rapidly with HOC1,'6strongly inhibited the reaction. DISCUSSION We have demonstrated that human neutrophils stimulated with opsonized zymosan promote the loss of MCD by a process that depends on myeloperoxidase, H,O, and CI-. Previous studies with the purified enzyme4.'' have shown that HOCl is the species responsible for this reaction. Our results are consistent with this and show that extracellularly generated HOCl promotes the loss of MCD by neutrophils stimulated with opsonized zymosan. A novel finding in this investigation is that production of HOCl by neutrophils can be inhibited by SOD. At low rates of oxidant production SOD did not inhibit the loss of MCD. However, with increasing oxidant production, SOD inhibited the loss of MCD by up t&-70%. Although inhibition by SOD and catalase could implicate -OH, this radical was excluded because the loss of MCD was dependent on myeloperoxidase and was unaffected by -OHscavengers or inhibitors of the Fenton reaction. Our results indicate that neutrophils can utilize 0; to enhance their extracellular production of HOCI. The striking similarity between the effects of 0; on HOCl production by purified myeloperoxidase' and neutrophils stimulated with opsonized zymosan (Figure 2), suggest that the mechanism established for the purified enzyme (Figure 3) is operative in neutrophils. 0; enhanced production of HOCI because i t prevents inactive compound I1 from accumulating. Chlorination occurs in the presence of a flux of 0;. even though 0; promotes formation of compound 111, because this form of myenoci



Compound I

Compound II

FIGURE 3. The mechanism of myeloperoxidase-dependentchlorination in the presence of superoxide (7).


flux of

Free Radic Res Downloaded from by Chulalongkorn University on 12/31/14 For personal use only.



loperoxidase reacts with H 2 0 2to give compound II, which is then reduced to active ferric myeloperoxidase by 0;. SOD should inhibit production of HOCl by neutrophils only under conditions where compound II accumulates and its turnover is limiting. Agents other than O;, such as ascorbate'* and urate' can reduce compound II. When they are present, the inhibitory effect of SOD will be masked. However, the high flux of 0;produced by neutrophils should ensure that 0; is the major reductant in vivo. In this investigation the MCD used to detect HOCl also promotes the accumulation of compound lI.l9 However, we have recently shown that high concentrations of H 2 0 2 inhibit myeloperoxidase by promoting the formation of compound 11, as do tryptophan and serum components in the presence of H,02.'9 This inhibition is prevented by a flux of 0; .* An important conclusion arising from this investigation is that SOD and catalase can inhibit reactions of neutrophils that are not due to .OH.This is especially relevant since recent studies have shown that isolated neutrophils do not produce -OH,'' and that myeloperoxidase is an effective inhibitor of -OH production.*' *OH has been suggested as the damaging agent in a wide variety of inflammatory conditions, as well as in neutrophil self-destruction,** bacterial killing by neutrophils," and Cundidu killing by monocytes2' because SOD and catalase inhibited these processes. Inhibition of 0;-driven HOCl production by both enzymes, as observed in the present study, provides an alternative explanation for these results. Hence, these results imply that 0; may potentiate inflammatory tissue damage by enhancing the production of HOCI, and that the anti-inflammatory effect of SOD may, in part, be due to the inhibition of this reaction. Acknowledgement This work was supported by the Medical Research Council of New Zealand.

References I . S.J. Weiss and A.F. LoBuglio (1982) Phagocyte-generated oxygen metabolites and cellular injury. Laborarory Invesrigurion. 47, 5-1 8. 2. S.J. Weiss, R. Klein, A. Slivka and M. Wei (1982) Chlorination of taurine by human neutrophils. Evidence for hypochlorous acid generation. Journal of Clinical Invesrigarion. 70, 598607. 3. C.S. Footc, T.E. Goyne and R.I. Lehrer (1953) Assessment of chlorination by human neutrophils. Nature. 301, 715-716. 4. J.E. Harrison and J. Schultz (1976) Studies on the chlorinating activity of myeloperoxidase. Jntrrnnl 01Biological Cheniisrrv. 251, 1371-1374. 5 . R. Makino, T. Tanaka. T. lizuka, Y. Ishimura and S. Kancgasaki (1986) Stoichiomctric conversion

6. 7. 8.



of oxygen to superoxide anion during the respiratory burst in neutrophils. Journal of Biological Chemistry. 261, 11444-1 1447. Y. Odajima and 1. Yamazaki (1972) Myeloperoxidase of the leukocytes of normal blood. 111 The reaction of ferric myeloperoxidase with superoxide anion. Biuchimicu Biuphysicu Actu. 284, 355-359. A.J. Kettle and C.C. Winterbourn (1988) Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid. Bioclrcniicul Journal. 252. 529-536. A.J. Kettle and C.C. Winterbourn (1989) The influence of superoxide on the kinetics of myeloperoxidase measured with a H,O, electrode. Bioclim~icalJournal. 263, 823-828. C.C. Winterbourn, R. Garcia and A.W. Scgal (1985) Production of the superoxide adduct of myeloperoxidase (compound 111) by stimulated neutrophils and its reactivity with hydrogen peroxide and chloride. Biocliemicul Juurnal. 228, 583-592. R.A. Clark. P.J. Stone A. El-hag. J.D. Calore and C. Franzblau (1981) Myeloperoxidasecatalysed


A.J. KETTLE AND C.C. WINTERBOURN inactivation of a, -protease inhibitor by human neutrophils. The Journal of Biological Chemistry. 256, 3348-3353.

II. 12. 13. 14.

Free Radic Res Downloaded from by Chulalongkorn University on 12/31/14 For personal use only.

15. 16. 17. 18. 19. 20.

A.J. Kettle and C.C. Winterbourn (1990) Superoxide enhances the production of hypochlorous acid by stimulated human neutrophils. BBA, In Press. A. Boyum (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation; and of granulocytes by combining centrifugation and sedimentation at Ig. Scandinavian Journal of Clinical Investigation (Suppl.) 21, 77-89. T. Odajima and I. Yamazaki (1970) Myeloperoxidase of the leukocytes of normal blood. 1. Reaction of myeloperoxidase with hydrogen peroxide. Biochimica Biophysica Acla. 206, 7 1-77. L.P. Hager. D.R. Morris, F.S.Brown and H. Ebenvein (1966) Chloroperoxidase. II. Utilization of halogen ions. The Journal of Biological Chemisrry. 241, 1769-1 777. B. Halliwell and J.M.C. Gutteridge (1985) Free radicals in human disease. Molecular Aspects of Medicine. 8, 89- 193. M.F. Tsan (1982) Myeloperoxidase-mediatedoxidation of methionine and amino acid decarboxylation. Infection and Immunisation, 36, 136-141. C.C. Winterbourn (1985) Comparative reactivities of various biological compounds with myeloperoxidase-hydrogen peroxidechloride, and similarity of the oxidant to hypochlorite. Biochiniica Biophysica Acra, 840,204-210. B.G.J.M. Bolscher, G.R. Zoutberg, R.A. Cuperus and R. Wever (1984) Vitamin C stimulates the chlorinating activity of human myeloperoxidase. Bioc/rinticu Biopliysicu Actu. 784, 189-191. A.J. Kettle and C.C. Winterbourn (1988) The mechanism of myeloperoxidase-dependentchlorination of monochlorodimedon. Biochimica Biophy.rico Acra. 957, 185-181. M.S. Cohen. B.E. Britigan, D.J. Hassett and G.M. Rosen (1988) Do human neutrophils form hydroxyl radical? Evaluation of an unresolved controversy. Free Radicul Biological Medicine, 5, 81-88.

C.C. Winterbourn (1986) Myeloperoxidase as an effective inhibitor of hydroxyl radical production. The Journal of Clinical Invesligalion. 18, 545-550. 22. M.L. Salin and J.M. McCord (1975) Free radicals and inflammation: Protection of phagocytosing leukocytes by superoxide disrnutase. The Journal of Clinical Invesrigarion. 56, 13 19-1323. 23. R.B. Johnston Jr., B.B. Keele Jr., H. Misra, J.E. Lehmeyer, L.S. Webb, R.L. Haehner and K.V. Rajagopalan (1975) The role of superoxide anion generation in phagocytic bactericidial activity. Studies with normal and chronic granulomatous disease leukocytes. The Journal of Clinical Invesligalion. 55, 1357-1372. 24. M. Sasada, A. Kubo, T. Nishimura, T. Kakita. T. Moriguchi. K. Yamamoto and H. Uchino (1987) Canadacidal activity of monocyte-derived human niacrophages. Relationship between candidal killing and oxygen radical generation by human macrophages. Journal 9 / Lcukocyrc Biology, 41, 21.


Accepted by Prof. G. Czapski

The influence of superoxide on the production of hypochlorous acid by human neutrophils.

Human neutrophils stimulated with opsonized zymosan promoted hypochlorous acid (HOCl)-dependent loss of monochlorodimedon. Formation of HOCl was compl...
352KB Sizes 0 Downloads 0 Views